Funded projects

Nutritional requirements of fish cell lines – development of a serum-free culture medium (L-15Plus)Manmade chemicals have become an essential commodity in our daily lives, from personal care to medicine and agriculture. Regulators require companies to conduct safety testing because, despite their benefits, such chemicals may also pose risks to human and environmental health. Environmental risk assessments still heavily rely on tests with fish. To replace such fish tests, we are developing strategies and assay procedures based on permanent fish cell lines of rainbow trout (Oncorhynchus mykiss), a key species for environmental protection. The most advanced procedure is the RTgill-W1 cell line assay to predict fish acute toxicity, which has already undergone several validation steps; it has recently been accepted as ISO guideline (21115) and been included in the OECD test guideline development programme. Yet, while these advances pave the way to replace fish, the routine culture of these cells and certain chemical exposure assays still require fetal calf serum to grow the cells. It is now time to eliminate the need of this last animal-derived component for fish cell culture and to move not only to fish- but rather to completely animal-free testing. To achieve this aim, we propose a systematic screening approach that will allow us to rapidly test and identify essential nutritional factors for fish cell proliferation. The broadly used and well accepted fish cell culture medium, Leibovitz’ L-15, will be used as a base and supplemented to obtain a serum- and animal-component free medium: L-15Plus.

Rodents have a right to best surgical practiceSurgery is an integral part of many experimental animal studies. Anaesthesia and analgesia protocols are obvious targets for refinement. Good surgical practice, however, is often neglected. Performing surgery fast, minimally invasive and with optimal peri-and post-operative care helps reduce animal suffering, enables faster recovery with fewer complications and improves the reproducibility of study results. For human and clinical veterinary surgery, it is state of the art to use aseptic technique and good peri-and postoperative care to alleviate the impact of a surgical intervention and to prevent postoperative complications. Controversially, rodent surgeons rarely adhere to these principles. There seem to be a common misconception that this is unnecessary in rodents. Even though the legal requirements for rodent and large animals are the same, it seems that researchers performing surgery on rodents often have limited medical or veterinary training and experience compared to large animal surgeons. There currently exists no generally accepted guidelines on experimental surgery in Switzerland. We think that there is an urgent need to refine the current surgical practice in rodents. Therefore, we aim to develop minimal standards for training and conduction of rodent surgery through a stepwise approach. First, we will conduct a systematic review of the available literature for the currently applied standards for experimental surgery. We will use an online survey and interviews to evaluate the current practice used by researchers performing rodent surgery to identify gaps. Based on the results we will develop a catalogue of measures involving key opinion leaders, a framework of best practice guidelines, and training modules for researchers.

Breeding management software for genetically modified rodents Scientists frequently use genetically modified animals in basic and applied research. They often combine multiple genetic modifications requiring complex breeding schemes. Some of the surplus animals that do not carry the required traits cannot be used for research or further breeding and are usually euthanized. Such supernumerary animals cannot be entirely prevented, but their numbers must be kept to the minimum possible. Therefore, we aim to optimise breeding strategies for complex genetic models, which is not possible manually. There is currently no software on the market to support us to better plan our breeding strategies. We aim to develop a stand-alone software tool, which will allow researchers to identify the best breeding strategies and thereby reduce the number of surplus animals. This is especially important when multiple traits are combined and the Mendelian laws lead to birth of surplus animals. The application of the software will reduce surplus animals to the essential minimum.

Recombinant laminin-like proteins for organoid cultures free of animal-derived basement membrane extractOrganoids are miniature organs generated from stem cells that provide unique in vitro models of organs in health and disease, and hold promises for personalized medicine and tissue engineering. They alleviate the need for animal experiments, as they enable the study of biomedical questions directly in vitro. They have become a cornerstone of 21st century biomedical research, and they still constitute a fast-developing field. In many cases, the tissue responsible for the organ-specific function – the parenchyma – is an epithelium, supported by a specialized matrix called a basement membrane. Examples include the intestine, liver, pancreas, kidney, lung, blood vessels, and skin. The brain also develops from neuroepithelium. Researchers have relied on basement membrane extract (BME) as a 3D matrix for their organoid cultures. BME is currently prepared from so-called Engelbreth-Holm-Swarm (EHS) tumours grown subcutaneously in mice. There is a strong need to develop BME alternatives, which avoid the use of mice, while simultaneously facilitating upscaling and clinical translation. We previously introduced alternative hydrogels that provide optimal physical properties: one based on the synthetic polymer poly(ethylene glycol) (PEG) and another based on the surgical tissue sealant fibrin Unfortunately, one last critical bioactive component, laminin, still needs to be purified from the BME, and has proven very challenging to replace. Here we propose to design and produce recombinant laminin fragments that retain the structure and function of the native protein while being easy to anchor to engineered matrices and amenable to mass-production.

A CRISPR/Cas9-screening platform to decipher conserved cell fate specification networks in vivoThe adult human body consists of hundreds, if not thousands, of distinct cell types. Examples include different neurons in the brain, epithelial cells lining the gut, or the cells that produce the bone or cartilage in our skeletons. During embryogenesis, all of these diverse cell types develop from a single progenitor cell: the fertilized egg. Understanding the different molecular mechanisms that orchestrate this diversification can help us re-create a particular cell type in a cell culture dish. Having access to such ‘in vitro’-generated cells would then allow researchers to use them in tissue repair and replacement strategies in human patients. What are the potential difficulties in achieving these goals? Many cell type specification processes require complex interactions with the surrounding tissue. Hence, only in the context of a developing embryo can these processes be fully understood; making the use of non-human ‘model organisms’ indispensable. This, however, raises additional questions: how can we minimize the number of experimental animals used in these studies; and how can we ensure that findings in such model organisms also translate to us humans? To address these challenges, here we propose to integrate comparative genomics data to define conserved ‘core regulatory switches’ that specify a given cell type across species. We will then functionally test the relevance of these candidate ‘switches’ using genetic perturbations in chicken embryos. Our experimental approach will prevent the euthanasia of any pregnant female animal while at the same time maximize its relevance for the subsequent in vitro specification of human cell types.

3D heart models for cardiac surgery training Surgery training on live animals is a controversial subject. While prohibited in Switzerland, surgical training on live animals is performed in many other countries. Several scientific publications as well as organisers of workshops open to a large audience of surgeons support this practice. Our goal is to develop a realistic and cost-effective alternative to the use of live animals for cardiovascular training. This project aims to first impart a segmentation procedure from CT and MRI data based on open-source software and secondly to develop a method for realistic 3D prints of congenital heart diseases based on rubber silicone. We will then validate the utility of this 3D printed hearts models by cardiovascular surgery training and write a paper to disseminate our protocol in order to motivate other countries to replace live animals for surgical training by these 3D printed models.